Novel motor proteins and methods for their use

ABSTRACT

The invention provides isolated nucleic acid and amino acid sequences of HsKif9, antibodies to HsKif9, methods of screening for HsKif9 modulators using biologically active HsKif9, and kits for screening for HsKif9 modulators.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. Ser. No.09/634,957, filed Aug. 8, 2000, which is incorporated herein in itsentirety for all purposes.

FIELD OF THE INVENTION

The invention provides isolated nucleic acid and amino acid sequences ofHsKif9, methods of detecting HsKif9 and screening for HsKif9 modulatorsusing biologically active HsKif9, and kits for screening for HsKif9modulators.

BACKGROUND OF THE INVENTION

The kinesin superfamily is an extended family of related microtubulemotor proteins. It can be classified into at least eight subfamiliesbased on primary amino acid sequence, domain structure, velocity ofmovement, and cellular function. This family is exemplified by “true”kinesin, which was first isolated from the axoplasm of squid, where itis believed to play a role in anterograde axonal transport of vesiclesand organelles (see, e.g., Goldstein, Anne Rev. Genet. 27:319-351(1993)). Kinesin uses ATP to generate force and directional movementassociated with microtubules.

Within this functional group of kinesins resides a group of kinesinsfrom several organisms that share significant sequence homology,including mouse kinesin Kif9. See, Nakagawa et al. (1997) Proc. Natl.Acad. Sci. USA 94:96549659; and GenBank Accession Number AB001434, eachof which is incorporated herein by reference for all purposes.

The discovery of a new kinesin motor protein which is the human orthologof mouse kinesin Kif9, and the polynucleotides encoding it satisfies aneed in the art by providing new compositions which are useful in thediagnosis, prevention, and treatment of cancer, neurological disorders,and disorders of vesicular transport.

SUMMARY OF THE INVENTION

The present invention is based on the discovery of a new human kinesinmotor protein, HsKif9, the polynucleotide encoding HsKif9, and the useof these compositions for the diagnosis, treatment, or prevention ofcancer, neurological disorders, and disorders of vesicular transport.

In one aspect, the invention provides an isolated nucleic acid sequenceencoding a kinesin superfamily motor protein, wherein the motor proteinhas the following properties: (i) the protein's activity includesmicrotubule stimulated ATPase activity; and (ii) the protein has asequence that has greater than 70% amino acid sequence identity to SEQID NO:2 or SEQ ID NO:4 as measured using a sequence comparisonalgorithm. In one embodiment, the protein further specifically binds topolyclonal antibodies raised against SEQ ID NO:2 or SEQ ID NO:4.

In one embodiment, the nucleic acid encodes HsKif9 or a fragmentthereof. In another embodiment, the nucleic acid encodes SEQ ID NO:2 orSEQ ID NO:4. In another embodiment, the nucleic acid has a nucleotidesequence of SEQ ID NO: 1 or SEQ ID NO:3.

In one aspect, the nucleic acid comprises a sequence which encodes anamino acid sequence which has greater than 70% sequence identity withSEQ ID NO:2 or SEQ ID NO:4, preferably greater than 80%, more preferablygreater than 90%, more preferably greater than 95% or, in anotherembodiment, has 98 to 100% sequence identity with SEQ ID NO:2 or SEQ IDNO:4.

In one embodiment, the nucleic acid comprises a sequence which hasgreater than 55 or 60% sequence identity with SEQ ID NO: 1 or SEQ IDNO:3, preferably greater than 70%, more preferably greater than 80%,more preferably greater than 90 or 95% or, in another embodiment, has 98to 100% sequence identity with SEQ ID NO: 1 or SEQ ID NO:3. In anotherembodiment provided herein, the nucleic acid hybridizes under stringentconditions to a nucleic acid having a sequence or complementary sequenceof SEQ ID NO:1 or SEQ ID NO:3.

In another aspect, the invention provides an expression vectorcomprising a nucleic acid encoding a kinesin superfamily motor protein,wherein the motor protein has the following properties: (i) theprotein's activity includes microtubule stimulated ATPase activity; and(ii) the protein has a sequence that has greater than 70% amino acidsequence identity to SEQ ID NO:2 or SEQ ID NO:4 as measured using asequence comparison algorithm. The invention further provides a hostcell transfected with the vector.

In another aspect, the invention provides an isolated kinesinsuperfamily motor protein, wherein the protein has one or more of theproperties described above. In one embodiment, the protein specificallybinds to polyclonal antibodies generated against a motor domain, taildomain or other fragment of HsKif9. In another embodiment, the proteincomprises an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.

In one aspect, the protein provided herein comprises an amino acidsequence which has greater than 70% sequence identity with SEQ ID NO:2or SEQ ID NO:4, preferably greater than 80%, more preferably greaterthan 90%, more preferably greater than 95% or, in another embodiment,has 98 to 100% sequence identity with SEQ ID NO:2 or SEQ ID NO:4.

The invention features a substantially purified polypeptide comprisingthe amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 or a fragmentthereof and more particularly, the motor domain of the amino acidsequence of SEQ ED NO:2 or a fragment thereof, such as SEQ ID NO:4.

In one embodiment, the present invention provides a method ofidentifying a candidate agent as a modulator of the activity of a targetprotein. The method comprises adding a candidate agent to a mixturecomprising a target protein which directly or indirectly produces ADP orphosphate, under conditions that normally allow the production of ADP orphosphate. The method further comprises subjecting the mixture to areaction that uses said ADP or phosphate as a substrate under conditionsthat normally allow the ADP or phosphate to be utilized and determiningthe level of activity of the reaction as a measure of the concentrationof ADP or phosphate. A change in the level between the presence andabsence of the candidate agent indicates a modulator of the targetprotein.

The phrase “se ADP or phosphate” means that the ADP or phosphate aredirectly acted upon by detection reagents. In one case, the ADP, forexample, can be hydrolyzed or can be phosphorylated. As another example,the phosphate can be added to another compound. As used herein, in eachof these cases, ADP or phosphate is acting as a substrate.

Preferably, the target protein either directly or indirectly producesADP or phosphate and comprises a motor domain. More preferably, thetarget protein comprises a kinesin superfamily motor protein asdescribed above and most preferably, the target protein comprises HsKif9or a fragment thereof.

Also provided are modulators of the target protein including agents forthe treatment of cellular proliferation, including cancer, hyperplasias,restenosis, cardiac hypertrophy, immune disorders and inflammation. Theagents and compositions provided herein can be used in variety ofapplications which include the formulation of sprays, powders, and othercompositions. Also provided herein are methods of treating cellularproliferation disorders such as cancer, hyperplasias, restenosis,cardiac hypertrophy, immune disorders and inflammation, for treatingdisorders associated with HsKif9 activity, and for inhibiting HsKif9.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show an embodiment of a nucleic acid sequence encodingHsKif9 (SEQ ID NO:1).

FIG. 2 shows the amino acid sequence of HsKif9 shown in FIG. 1 (SEQ IDNO:2).

FIG. 3 shows a nucleic acid sequence encoding a motor domain fragment ofHsKif9 (SEQ ID NO:3).

FIG. 4 shows the amino acid sequence of the motor domain fragment shownin FIG. 3 (SEQ ID NO:4).

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

“ADP” refers to adenosine diphosphate and also includes ADP analogs,including, but not limited to, deoxyadenosine diphosphate (dADP) andadenosine analogs.

“Antibody” refers to a polypeptide substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof whichspecifically bind and recognize an analyte (antigen). The recognizedimmunoglobulin genes include the kappa, lambda, alpha, gamma, delta,epsilon and mu constant region genes, as well as the myriadimmunoglobulin variable region genes. Light chains are classified aseither kappa or lambda. Heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes, IgG,IgK, IgA, Ig)D and IgE, respectively. The term antibody also includesantibody fragments either produced by the modification of wholeantibodies or those synthesized de novo using recombinant DNAmethodologies.

An “anti-HsKif9” antibody is an antibody or antibody fragment thatspecifically binds a polypeptide encoded by the HsKif9 gene, cDNA, or asubsequence thereof

“Biologically active” target protein refers to a target protein that hasone or more of kinesin protein's biological activities, including, butnot limited to microtubule stimulated ATPase activity, as tested, e.g.,in an ATPase assay. Biological activity can also be demonstrated in amicrotubule gliding assay or a microtubule binding assay. “ATPaseactivity” refers to ability to hydrolyze ATP. Other activities includepolymerization/depolymerization (effects on microtubule dynamics),binding to other proteins of the spindle, binding to proteins involvedin cell-cycle control, or serving as a substrate to other enzymes, suchas kinases or proteases and specific kinesin cellular activities, suchas chromosome congregation, axonal transport, etc.

“Biological sample” as used herein is a sample of biological tissue orfluid that contains a target protein or a fragment thereof or nucleicacid encoding a target protein or a fragment thereof. Biological samplesmay also include sections of tissues such as frozen sections taken forhistological purposes. A biological sample comprises at least one cell,preferably plant or vertebrate. Embodiments include cells obtained froma eukaryotic organism, preferably eukaryotes such as fungi, plants,insects, protozoa, birds, fish, reptiles, and preferably a mammal suchas rat, mice, cow, dog, guinea pig, or rabbit, and most preferably aprimate such as chimpanzees or humans.

A “comparison window” includes reference to a segment of any one of thenumber of contiguous positions selected from the group consisting offrom 25 to 600, usually about 50 to about 200, more usually about 100 toabout 150 in which a sequence may be compared to a reference sequence ofthe same number of contiguous positions after the two sequences areoptimally aligned. Methods of alignment of sequences for comparison arewell-known in the art. Optimal alignment of sequences for comparison canbe conducted, e.g., by the local homology algorithm of Smith & Waterman,Adv. Appl. Math. 2:482 (1981), by the global alignment algorithm ofNeedleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search forsimilarity methods of Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444 (1988) and Altschul et al. Nucleic Acids Res. 25(17): 3389-3402(1997), by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and BLAST in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manualalignment and visual inspection (see, e.g., Ausubel et al., supra).

This algorithm involves first identifying high scoring sequence pairs(HSPs) by identifying short words of length W in the query sequence,which either match or satisfy some positive-valued threshold score Twhen aligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are thenextended in both directions along each sequence for as far as thecumulative alignment score can be increased. Cumulative scores arecalculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always >0) and N (penalty scorefor mismatching residues; always,)). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. For identifying whether a nucleic acid or polypeptide is withinthe scope of the invention, the default parameters of the BLAST programsare suitable. The BLASTN program (for nucleotide sequences) uses asdefaults a word length (W) of 11, an expectation (E) of 10, M=5, N=4,and a comparison of both strands. For amino acid sequences, the BLASTPprogram uses as defaults a word length (W) of 3, an expectation (F) of10, and the BLOSUM62 scoring matrix. The TBLATN program (using proteinsequence for nucleotide sequence) uses as defaults a word length (W) of3, an expectation (L) of 10, and a BLOSUM 62 scoring matrix. See,Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad.Sci. USA 90:5873-5787). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

Another example of a useful algorithm is PILEUP. PILEUP creates amultiple sequence alignment from a group of related sequences usingprogressive, pairwise alignments. It can also plot a dendrogram showingthe clustering relationships used to create the alignment. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle,J. Mol. Evol. 35:351-360 (1987). The method used is similar to themethod described by Higgins & Sharp, CABIOS 5:151-153 (1989). As ageneral rule, PileUp can align up to 500 sequences, with any singlesequence in the final alignment restricted to a maximum length of 7,000characters.

The multiple alignment procedure begins with the pairwise alignment ofthe two most similar sequences, producing a cluster of two alignedsequences. This cluster can then be aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences canbe aligned by a simple extension of the pairwise alignment of twoindividual sequences. A series of such pairwise alignments that includesincreasingly dissimilar sequences and clusters of sequences at eachiteration produces the final alignment.

“Variant” applies to both amino acid and nucleic acid sequences. Withrespect to particular nucleic acid sequences, conservatively modifiedvariants refers to those nucleic acids which encode identical oressentially identical amino acid sequences, or where the nucleic aciddoes not encode an amino acid sequence, to essentially identicalsequences.

Because of the degeneracy of the genetic code, a large number offunctionally identical nucleic acids encode any given protein. Forinstance, the codons GCA, GCC, GCG and GCT all encode the amino acidalanine. Thus, at every position where an alanine is specified by acodon, the codon can be altered to any of the corresponding codonsdescribed without altering the encoded polypeptide. Such nucleic acidvariations are “silent variations,” which are one species ofconservatively modified variations. Every nucleic acid sequence hereinwhich encodes a polypeptide also describes every possible silentvariation of the nucleic acid. One of skill will recognize that eachdegenerate codon in a nucleic acid can be modified to yield afunctionally identical molecule.

Accordingly, each silent variation of a nucleic acid which encodes apolypeptide is implicit in each described sequence. Also included withinthe definition of target proteins of the present invention are aminoacid sequence variants of wild-type target proteins. These variants fallinto one or more of three classes: substitutional, insertional ordeletional variants. These variants ordinarily are prepared by sitespecific mutagenesis of nucleotides in the DNA encoding the targetprotein, using cassette or PCR mutagenesis or other techniques wellknown in the art, to produce DNA encoding the variant, and thereafterexpressing the DNA in recombinant cell culture. Variant target proteinfragments having up to about 100-150 amino acid residues may be preparedby in vitro synthesis using established techniques. Amino acid sequencevariants are characterized by the predetermined nature of the variation,a feature that sets them apart from naturally occurring allelic orinterspecies variation of the target protein amino acid sequence. Thevariants typically exhibit the same qualitative biological activity asthe naturally occurring analogue, although variants can also be selectedwhich have modified characteristics.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about 1 to about 20 amino acids,although considerably longer insertions may be tolerated. Deletionsrange from about 1 to about 20 residues, although in some cases,deletions may be much longer.

Substitutions, deletions, and insertions or any combinations thereof maybe used to arrive at a final derivative. Generally, these changes aredone on a few amino acids to minimize the alteration of the molecule.However, larger characteristics may be tolerated in certaincircumstances.

The following six groups each contain amino acids that are conservativesubstitutions for one another:

-   1) Alanine (A), Serine (S), Threonine (I);-   2) Aspartic acid (1), Glutamic acid ( );-   3) Asparagine (N), Glutamine (Q);-   4) Arginie (R), Lysine (K);-   5) Isoleucine (1), Leucine (L), Methionine (M), Valine (V); and-   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).    (see, e.g., Creighton, Proteins (1984)).

“Cytoskeletal component” denotes any molecule that is found inassociation with the cellular cytoskeleton, that plays a role inmaintaining or regulating the structural integrity of the cytoskeleton,or that mediates or regulates motile events mediated by thecytoskeleton. Cytoskeletal component is intended to include cytoskeletalpolymers (e.g., actin filaments, microtubules, intermediate filaments,myosin fragments), molecular motors (e.g., kinesins, myosins, dyneins),cytoskeleton associated regulatory proteins (e.g., tropomysin,alpha-actinin) and cytoskeletal associated binding proteins (e.g.,microtubules associated proteins, actin binding proteins).

“Cytoskeletal function” refers to biological roles of the cytoskeleton,including but not limited to the providing of structural organization(e.g., microvilli, mitotic spindle) and the mediation of motile eventswithin the cell (e.g., muscle contraction, mitotic chromosome movements,contractile ring formation and function, pseudopodal movement, activecell surface deformations, vesicle formation and translocation)

A “diagnostic” as used herein is a compound, method, system, or devicethat assists in the identification and characterization of a health ordisease state. The diagnostic can be used in standard assays as is knownin the art.

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

“High stringency conditions” may be identified by those that: (1) employlow ionic strength and high temperature for washing, for example 0.015 Msodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at50° C.; (2) employ during hybridization a denaturing agent such asformamide, for example, 50% (v/v) formamide with 0.1% bovine serumalbumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphatebuffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodiumcitrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS,and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC(sodium chloride/sodium citrate) and 50% formamide at 55° C., followedby a high-stringency wash consisting of 0.1×SSC containing EDTA at 55°C.

“High throughput screening” as used herein refers to an assay whichprovides for multiple candidate agents or samples to be screenedsimultaneously. As further described below, examples of such assays mayinclude the use of microtiter plates which are especially convenientbecause a large number of assays can be carried out simultaneously,using small amounts of reagents and samples.

By “host cell” is meant a cell that contains an expression vector andsupports the replication or expression of the expression vector. Hostcells may be prokaryotic cells such as E. coli, or eukaryotic cells suchas yeast, insect, amphibian, or mammalian cells such as CHO, HeLa andthe like, or plant cells. Both primary cells and cultured cell lines areincluded in this definition.

The phrase “hybridizing specifically to” refers to the binding,duplexing, or hybridizing of a molecule only to a particular nucleotidesequence under stringent conditions when that sequence is present in acomplex mixture (e.g., total cellular) DNA or RNA. Stringent conditionsare sequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (T) for the specific sequence at adefined ionic strength and pH. The T_(m) is the temperature (underdefined ionic strength, pH, and nucleic acid concentration) at which 50%of the probes complementary to the target sequence hybridize to thetarget sequence at equilibrium. Typically, stringent conditions will bethose in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.05 to 1.0 M sodium ion concentration (or othersalts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. forshort probes (e.g., 10 to 50 nucleotides) and at least about 60° C. forlong probes (e.g., greater than 50 nucleotides). Stringent conditionsmay also be achieved with the addition of destabilizing agents such asformamide.

The terms “identical” or percent “identity”, in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence over a comparisonwindow, as measured using one of the following sequence comparisonalgorithms or by manual alignment and visual inspection. Preferably, thepercent identity exists over a region of the sequence that is at leastabout 25 amino acids in length, more preferably over a region that is 50or 100 amino acids in length. This definition also refers to thecomplement of a test sequence, provided that the test sequence has adesignated or substantial identity to a reference sequence. Preferably,the percent identity exists over a region of the sequence that is atleast about 25 nucleotides in length, more preferably over a region thatis 50 or 100 nucleotides in length.

When percentage of sequence identity is used in reference to proteins orpeptides, it is recognized that residue positions that are not identicaloften differ by conservative amino acid substitutions, where amino acidresidues are substituted for other amino acid residues with similarchemical properties (e.g,. charge or hydrophobicity) and therefore donot change the functional properties of the molecule. Where sequencesdiffer in conservative substitutions, the percent sequence identity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well known to thoseof skill in the art The scoring of conservative substitutions can becalculated according to, e.g., the algorithm of Meyers & Millers,Computer Applic. Biol. Sci. 4:11-17 (1988), e.g., as implemented in theprogram PC/GENE ([ntelligenetics, Mountain View, Calif.).

The terms “isolated”, “purified”, or “biologically pure” refer tomaterial that is substantially or essentially free from components whichnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein that is the predominantspecies present in a preparation is substantially purified. In anisolated gene, the nucleic acid of interest is separated from openreading frames which flank the gene of interest and encode proteinsother than the protein of interest. The term “purified” denotes that anucleic acid or protein gives rise to essentially one band in anelectrophoretic gel. Particularly, it means that the nucleic acid orprotein is at least 85% pure, more preferably at least 95% pure, andmost preferably at least 99% pure.

A “label” is a composition detectable by spectroscopic, photochemical,biochemical, immunochemical, or chemical means. For example, usefullabels include fluorescent proteins such as green, yellow, red or bluefluorescent proteins, radioisotopes such as ³²P, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin, digoxigenin, or haptens and proteins for which antisera ormonoclonal antibodies are available (e.g., the polypeptide of SEQ IDNO:2 can be made detectable, e.g., by incorporating a radio-label intothe peptide, and used to detect antibodies specifically reactive withthe peptide).

A “labeled nucleic acid probe or oligonucleotide” is one that is bound,either covalently, through a linker, or through ionic, van der Waals, orhydrogen bonds to a label such that the presence of the probe may bedetected by detecting the presence of the label bound to the probe.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent than those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextransulfate, and 20 g/mL denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

“Modulators,” “iniibitors,” and “activators of a target protein” referto modulatory molecules identified using in vitro and in vivo assays fortarget protein activity. Such assays include ATPase activity,microtubule gliding, microtabule depolymerizing activity, and bindingactivity such as microtubule binding activity or binding of nucleotideanalogs. Samples or assays that are treated with a candidate agent at atest and control concentration. The control concentration can be zero.If there is a change in target protein activity between the twoconcentrations, this change indicates the identification of a modulator.A change in activity, which can be an increase or decrease, ispreferably a change of at least 20% to 50%, more preferably by at least50% to 75%, more preferably at least 75% to 100%, and more preferably150% to 200%, and most preferably is a change of at least 2 to 10 foldcompared to a control. Additionally, a change can be indicated by achange in binding specificity or substrate.

“Molecular motor” refers to a molecule that utilizes chemical energy togenerate mechanical force. According to one embodiment, the molecularmotor drives the motile properties of the cytoskeleton.

The phrase “motor domain” refers to the domain of a target protein thatconfers membership in the kinesin superfamily of motor proteins througha sequence identity of approximately 35-45% identity to the motor domainof true kinesin

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form. Unless specifically limited, the term encompassesnucleic acids containing known analogues of natural nucleotides whichhave similar binding properties as the reference nucleic acid and aremetabolized in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences as well asthe sequence explicitly indicated For example, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260)2605-2608(1985); Cassol et al. 1992; Rossolini et al. Mol. Cell. Probes 8:91-98(1994)). The term nucleic acid is used interchangeably with gene, cDNA,and mRNA encoded by a gene.

“Nucleic acid probe or oligonucleotide” is defined as a nucleic acidcapable of binding to a target nucleic acid of complementary sequencethrough one or more types of chemical bonds, usually throughcomplementary base pairing, usually through hydrogen bond formation. Asused herein, a probe may include natural (i.e., A, G, C, or T) ormodified bases. In addition, the bases in a probe may be joined by alinkage other than a phosphodiester bond, so long as it does notinterfere with hybridization. Thus, for example, probes may be peptidenucleic acids in which the constituent bases are joined by peptide bondsrather than phosphodiester linkages. It will be understood by one ofskill in the art that probes may bind target sequences lacking completecomplementarity with the probe sequence depending upon the stringency ofthe hybridization conditions. The probes are preferably directly labeledwith isotopes, chromophores, lumiphores, chromogens, or indirectlylabeled such as with biotin to which a streptavidine complex may laterbind. By assaying for the presence or absence of the probe, one candetect the presence or absence of the select sequence or subsequence.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidues is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. A target protein comprises a polypeptide demonstrated to haveat least microtubule stimulated ATPase activity. Amino acids may bereferred to herein by either their commonly known three letter symbolsor by Nomenclature Commission. Nucleotides, likewise, may be referred toby their commonly accepted single-letter codes, i.e., the one-lettersymbols recommended by the IUPAC-IUB.

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA box element. A promoter also optionally includes distal enhancer orrepressor elements which can be located as much as several thousand basepairs from the start site of transcription. A “constitutive” promoter isa promoter that is active under most environmental and developmentalconditions. An “inducible” promoter is a promoter that is underenvironmental or developmental regulation. The term “operably linked”refers to a functional linkage between a nucleic acid expression controlsequence (such as a promoter, or array of transcription factor bindingsites) and a second nucleic acid sequence, wherein the expressioncontrol sequence directs transcription of the nucleic acid correspondingto the second sequence.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein in a heterogeneous population of proteinsand other biologics. Thus, under designated immunoassay conditions, thespecified antibodies bind to a particular protein at least two times thebackground and do not substantially bind in a significant amount toother proteins present in the sample. Specific binding to an antibodyunder such conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, antibodies raised toHsKif9 with the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 can beselected to obtain only those antibodies that are specificallyimmunoreactive with HsKif9 and not with other proteins, except forpolymorphic variants, orthologs, alleles, and closely related homologuesof HsKif9. This selection may be achieved by subtracting out antibodiesthat cross react with molecules, for example, such as C. elegans unc-104and human Kif1A. A variety of immunoassay formats may be used to selectantibodies specifically immunoreactive with a particular protein. Forexample, solid-phase ELISA immunoassays are routinely used to selectantibodies specifically immunoreactive with a protein (see, e.g., Harlow& Lane, Antibodies, A Laboratory Manual (1988), for a description ofimmunoassay formats and conditions that can be used to determinespecific immunoreactivity). Typically a specific or selective reactionwill be at least twice background signal or noise and more typicallymore than 10 to 100 times background.

The phrase “selectively associates with” refers to the ability of anucleic acid to “selectively hybridize” with another as defined above,or the ability of an antibody to “selectively (or specifically) bind toa protein, as defined above.

“Test composition” (used interchangeably herein with “candidate agent”and “test compound” and “test agent”) refers to a molecule orcomposition whose effect on the interaction between one or morecytoskeletal components it is desired to assay. The “test composition”can be any molecule or mixture of molecules, optionally in a carrier.

A “therapeutic” as used herein refers to a compound which is believed tobe capable of modulating the cytoskeletal system in vivo which can haveapplication in both human and animal disease. Modulation of thecytoskeletal system would be desirable in a number of conditionsincluding, but not limited to: abnormal stimulation of endothelial cells(e.g., atherosclerosis), solid and hematopoetic tumors and tumormetastasis, benign tumors, for example, hemangiomas, acoustic neuromas,neurofibromas, pyogenic granulomas, vascular malfunctions, abnormalwound healing, inflammatory and immune disorders such as rheumatoidarthritis, Bechet's disease, gout or gouty arthritis, abnormalangiogenesis accompanying: rheumatoid arthritis, psoriasis, diabeticretinopathy, and other ocular angiogenic disesase such as, maculardegeneration, corneal graft rejection, corneal overgrowth, glaucoma, andOsler Webber syndrome.

II. The Target Protein

The present invention provides for the first time a nucleic acidencoding HsKif9. This protein is a member of the kinesin superfamily ofmotor proteins. More specifically, the HsKif9 sequence of FIG. 2 sharesapproximately 89% identity to a fragment of mouse Kif9. See Nakagawa etal. (1997) Proc. Natl. Acad. Sci. USA 94:9654-9.

In one aspect, HsKif9 can be defined by having at least one orpreferably more than one of the following functional and structuralcharacteristics. Functionally, HsKif9 will have microtubule-stimulatedATPase activity, and microtubule motor activity that is ATP dependentHsKif9 activity can also be described in terms of its ability to bindmicrotubules.

The novel nucleotides sequences provided herein encode HsKif9 orfragments thereof. Thus, in one aspect, the nucleic acids providedherein are defined by the novel proteins provided herein. The proteinprovided herein comprises an amino acid sequence which has one or moreof the following characteristics: greater than 70% sequence identitywith SEQ ID NO:2 or SEQ ID NO:4, preferably greater than 80%, morepreferably greater than 90%, more preferably greater than 95% or, inanother embodiment, has 98 to 100% sequence identity with SEQ ID NO:2 orSEQ ID NO:4. As described above, when describing the nucleotide in termsof SEQ ID NO:1 or SEQ ID NO:3, the sequence identity may be slightlylower due to the degeneracy in the genetic code.

The invention also includes fragments of the nucleotide sequence shownin FIG. 1 having at least 10, 15, 20, 25, 50, 100, 1000 or 2000contiguous nucleotides from SEQ ID NO:1 or a degenerate form thereof.Some fragments include the motor domain which occurs approximatelybetween positions four and 401 of the amino acid shown in FIG. 2(determined by sequence comparison of the motor domain of otherkinesins). Some such fragments can be used as hybridization probes orprimers. Unless otherwise apparent from the context, reference tonucleotide sequences shown in the Figures of sequence can refer to thesequence shown, its perfect complement or a duplex of the two strands.Also included within the definition of the target proteins are aminoacid sequence variants of wild-type target proteins.

Portions of the HsKif9 nucleotide sequence may be used to identifypolymorphic variants, orthologs, alleles, and homologues of HsKif9. Thisidentification can be made in vitro, e.g., under stringent hybridizationconditions and sequencing, or by using the sequence information in acomputer system for comparison with other nucleotide sequences. Sequencecomparison can be performed using any of the sequence comparisonalgorithms discussed below, with PILEUP as a preferred algorithm.

As will be appreciated by those in the art, the target proteins can bemade in a variety of ways, including both synthesis de novo and byexpressing a nucleic acid encoding the protein.

Target proteins of the present invention may also be modified in a wayto form chimeric molecules comprising a fusion of a target protein witha tag polypeptide which provides an epitope to which an anti-tagantibody can selectively bind. The epitope tag is generally placed atthe amino or carboxyl terminus of the target protein. Provision of theepitope tag enables the target protein to be readily detected, as wellas readily purified by affinity purification. Various tag epitopes arewell known in the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 (see, Field et al. (1988) Mol. Cell. Biol.8:2159); the c-myc tag and the 8F9, 3C7, 610, G4, B7 and 9E10 antibodiesthereto (see, Evans et al., (1985) Molecular and Cellular Biology,5:3610); and the Herpes Simplex virus glycoprotein D (gD) tag and itsantibody (see, Paborsky et al., (1990) Protein Engineering, 3:547).Other tag polypeptides include the Flag-peptide (see, Hopp et al. (1988)BioTechnology 6:1204); the KT3 epitope peptide (see, Martine et al.(1992) Science, 255:192); tubulin epitope peptide (see, Skinner (1991)J. Biol. Chem. 266:15173); and the T7 gene 10 protein peptide tag (see,Lutz-Freyermuth et al. (1990) Proc. Natl. Acad. Sci. USA 87:6393.

The biological activity of any of the peptides provided herein can beroutinely confirmed by the assays provided herein such as those whichassay ATPase activity or microtubule binding activity. In oneembodiment, polymorphic variants, alleles, and orthologs, homologues ofHsKif9 are confirmed by using a ATPase or microtubule binding assays asknown in the art.

The isolation of biologically active HsKif9 for the first time providesa means for assaying for modulators of this kinesin superfamily proteinBiologically active HsKif9 is useful for identifying modulators ofHsKif9 or fragments thereof and kinesin superfamily members using invitro assays such as microtubule gliding assays, ATPase assays (Kodamaet al., J. Biochem. 99:1465-1472 (1986); Stewart et al., Proc. Nat7Acad. Sci. USA 90:5209-5213 (1993)), and binding assays includingmicrotubule binding assays (Vale et al., Cell 42:39-50 (1985)). In vivoassays and uses are provided herein as well. Also provided herein aremethods of identifying candidate agents which bind to HsKif9 andportions thereof.

Some portions or fragments of HsKif9 include at least 7, 10, 15, 20, 35,50, 100, 250, 300, 350, 500, or 1000 contiguous amino acids from thesequence shown in FIG. 2. Some fragments contain fewer than 1000, 500,250, 100, or 50 contiguous amino acids from the sequence shown in FIG.2. For example, exemplary fragments include fragments having 15-50 aminoacids or 100-500 amino acids. Some fragments include a motor domain. Themotor domain runs from about amino acid 4 to about amino acid 401 of SEQID NO:1. Such fragments typically include this span, or an activeportion thereof. Some fragments include a ligand binding domain ofHsKif9. Nucleic acids encoding such fragments are also included in theinvention.

As further described herein, a wide variety of assays, therapeutic anddiagnostic methods are provided herein which utilize the novel compoundsdescribed herein. The uses and methods provided herein, as furtherdescribed below have in vivo, in situ, and in vitro applications, andcan be used in medicinal veterinary, agricultural and research basedapplications.

III. Isolation of the Gene Encoding HsKif9

A. General Recombinant DNA Methods

This invention relies on routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

For nucleic acids, sizes are given in either kilobases (cb) or basepairs (bp). These are estimates derived from agarose or acrylamide gelelectrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Proteins sizes are estimated from gelelectrophoresis, from mass spectroscopy, from sequenced proteins, fromderived amino acid sequences, or from published protein sequences.

Oligonucleotides that are not commercially available can be chemicallysynthesized according to the solid phase phosphoramidite triester methodfirst described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862(1981), using an automated synthesizer, as described in Van Devanter etal., Nucleic Acids Res. 12:6159-6168 (1984). Purification ofoligonucleotides is by either native acrylamide gel electrophoresis orby anion-exchange HPLC as described in Pearson. & Reanier, J. Chrom.225:137-149 (1983).

The sequence of the cloned genes and synthetic oligonucleotides can beverified after cloning using, e.g., the chain termination method forsequencing double-stranded templates of Wallace et al., Gene 16:21-26(1981).

B. Cloning Methods for the Isolation of Nucleotide Sequences Encoding

In general, the nucleic acid sequences encoding HsKif9 and relatednucleic acid sequence homologs are cloned from cDNA and genomic DNAlibraries or isolated using amplification techniques witholigonucleotide primers. Alternatively, expression libraries can be usedto clone HsKif9 and HsKif9 homologues by detected expressed homologuesimmunologically with antisera or purified antibodies made against HsKif9that also recognize and selectively bind to the HsKif9 homologue.Finally, amplification techniques using primers can be used to amplifyand isolate HsKif9 from DNA or RNA. Amplification techniques usingdegenerate primers can also be used to amplify and isolate HsKif9homologues. Amplification techniques using primers can also be used toisolate a nucleic acid encoding HsKif9. These primers can be used, e.g.,to amplify a probe of several hundred nucleotides, which is then used toscreen a library for full-length HsKif9.

Appropriate primers and probes for identifying the gene encodinghomologues of HsKif9 in other species are generated from comparisons ofthe sequences provided herein. As described above, antibodies can beused to identify HsKif9 homologues. For example, antibodies made to themotor domain of HsKif9 or to the whole protein are useful foridentifying HsKif9 homologues.

To make a cDNA library, one should choose a source that is rich in theMRINA of choice, e.g., HsKif9. For example, HsKif9 mRNA is overexpessedin testes. Expression is dramatically increased in testes and fetalliver. The mRNA is then made into cDNA using reverse transcriptase,ligated into a recombinant vector, and introduced into a recombinanthost for propagation, screening and cloning. Methods for making andscreening cDNA libraries are well known (see, e.g., Gubler & Hoffman,Gene 25: 263-269); Sambrook et al., supra; Ausubel et al., supra).

For a genomic library, the DNA is extracted from the tissue and eithermechanically sheared or enzymatically digested to yield fragments ofabout 12-20 kb. The fragments are then separated by gradientcentrifugation from undesired sizes and are constructed in bacteriophagelambda vectors. These vectors and phage are packaged in vitro.Recombinant phage are analyzed by plaque hybridization as described inBenton & Davis, Science 196:180-182 (1977). Colony hybridization is readout as generally described in Grunstein et al., Proc. Natl. Acad. Sci.USA, 72:3961-3965 (1975).

An alternative method of isolating HsKif9 nucleic acid and itshomologues combines the use of synthetic oligonucleotide primers andamplification of an RNA or DNA template (see U.S. Pat. Nos. 4,683,195and 4,683,202; PCR Protocols: A guide to Methods and Applications (Iiniset al., eds. 1990)). Methods such as polymerase chain reaction andligase chain reaction can be used to amplify nucleic acid sequences ofHsKif9 directly from mRNA, from cDNA, from genomic libraries or cDNAlibraries. Degenerate oligonucleotides can be designed to amplify HsKif9homologues using the sequences provided herein. Restriction endonucleasesites can be incorporated into the primers. Polymerase chain reaction orother in vitro amplification methods may also be useful for example, toclone nucleic acid sequences that code for proteins to be expressed, tomake nucleic acids to use as probes for detecting the presence of HsKif9encoding mRNA in physiological samples, for nucleic sequencing or forother purposes. Genes amplified by the PCR reaction can be purified fromagarose gels and cloned into an appropriate vector.

Gene expression of HsKif9 can also be analyzed by techniques known inthe art, e.g., reverse transcription and amplification of mRNA,isolation of total RNA or poly A+RNA, northern blotting, dot blotting,in situ hybridization, RNase protection, quantitative PCR, and the like.

Synthetic oligonucleotides can be used to construct recombinant HsKif9genes for use as probes or for expression of protein. This method isperformed using a series of overlapping oligonucleotides usually 40-120bp in length, representing both the sense and nonsense strands of thegene. These DNA fragments are then annealed, ligated and cloned.Alternatively, amplification techniques can be used with precise primersto amplify a specific subsequence of the HsKif9 gene. The specificsubsequence is then ligated into an expression vector.

The gene for HsKif9 is typically cloned into intermediate vectors beforetransformation into prokaryotic or eukaryotic cells for replicationand/or expression. The intermediate vectors are typically prokaryotevectors or shuttle vectors.

C. Expression in Prokaryotes and Eukaryotes

To obtain high level expression of a cloned gene, such as those cDNAsencoding HsKif9, it is important to construct an expression vector thatcontains a strong promoter to direct transcription, atranscription/translation terminator, and if for a nucleic acid encodinga protein, a ribosome binding site for translational initiation.Suitable bacterial promoters are well known in the art and described,e.g., in Sambrook et al. and Ausubel et al. Bacterial expression systemsfor expressing the HsKif9 protein are available in, e.g., E. colt,Bacillus sp., and Salmonella (Palva et al., Gene 22:229-235 (1983);Mosbach et al., Nature 302:543-545 (1983). Kits for such expressionsystems are commercially available. Eukaryotic expression systems formammalian cells, yeast, and insect cells are well known in the art andare also commercially available. The pET expression system (Novagen) isa preferred prokaryotic expression system.

The promoter used to direct expression of a heterologous nucleic aciddepends on the particular application. The promoter is preferablypositioned about the same distance from the heterologous transcriptionstart site as it is from the transcription start site in its naturalsetting. As is known in the art, however, some variation in thisdistance can be accommodated without loss of promoter function.

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the HsKif9 encodingnucleic acid in host cells. A typical expression cassette thus containsa promoter operably linked to the nucleic acid sequence encoding HsKif9and signals required for efficient polyadenylation of the transcript,ribosome binding sites, and translation termination. The nucleic acidsequence encoding HsKif9 may typically be linked to a cleavable signalpeptide sequence to promote secretion of the encoded protein by thetransformed cell Such signal peptides would include, among others, thesignal peptides from tissue plasminogen activator, insulin, and neurongrowth factor, and juvenile hormone esterase of Heliothis virescens.Additional elements of the cassette may include enhancers and, ifgenomic DNA is used as the structural gene, introns with functionalsplice donor and acceptor sites.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, pET23, and fusionexpression systems such as GST and LacZ. Epitope tags can also be addedto recombinant proteins to provide convenient methods of isolation,e.g., c-myc or histidine tags.

Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, cytomegalovirus vectors, papilloma virus vectors, and vectorsderived from Epstein Bar virus. Other exemplary eukaryotic vectorsinclude pMSG, pAVOO9/A⁺, pMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, andany other vector allowing expression of proteins under the direction ofthe SV40 early promoter, SV40 late promoter, CMV promoter,metallothionein promoter, murine mammary tumor virus promoter, Roussarcoma virus promoter, polyhedrin promoter, or other promoters showneffective for expression in eukaryotic cells.

Some expression systems have markers that provide gene amplificationsuch as thymidine kinase, hygromycin B phosphotransferase, anddihydrofolate reductase. Alternatively, high yield expression systemsnot involving gene amplification are also suitable, such as using abaculovirus vector in insect cells, with a HsKif9 encoding sequenceunder the direction of the polyhedrin promoter or other strongbaculovirus promoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of eukaryotic sequences. The particularantibiotic resistance gene chosen is not critical, any of the manyresistance genes known in the art are suitable. The prokaryoticsequences are preferably chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

Standard transfection or transformation methods are used to producebacterial, mammalian, yeast or insect cell lines that express largequantities of HsKif9 protein, which are then purified using standardtechniques (see, e.g., Colley et al., J. Biol. Chem. 264:17619-17622(1989); Guide to Protein Purification, in Methods in Enzymology , vol.182 (Ieutscher ed., 1990)).

Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques (see, e.g., Morrison, J. Bact.,132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology,101:347-362 (Wu et al., eds, 1983). Any of the well known procedures forintroducing foreign nucleotide sequences into host cells may be used.These include the use of calcium phosphate transfection, polybrene,protoplast fusion, electroporation, liposomes, microinjection, plasmavectors, viral vectors and any of the other well known methods forintroducing cloned genomic DNA, cDNA, synthetic DNA or other foreigngenetic material into a host cell (see, e.g. Sambrook et al., supra). Itis only necessary that the particular genetic engineering procedure usedbe capable of successfully introducing at least one gene into the hostcell capable of expressing HsKif9.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofHsKif9, which is recovered from the culture using standard techniquesidentified below.

IV. Purification of HsKi19 Protein

Either naturally occurring or recombinant HsKif9 can be purified for usein functional assays. In a preferred embodiment, the target proteins arepurified for use in the assays to provide substantially pure samples.Alternatively, the target protein need not be substantially pure as longas the sample comprising the target protein is substantially free ofother components that can contribute to the production of ADP orphosphate.

The target proteins may be isolated or purified in a variety of waysknown to those skilled in the art depending on what other components arepresent in the sample. Standard purification methods includeelectrophoretic, molecular, immunological, and chromatographictechniques, including ion exchange, hydrophobic, affinity, andreverse-phase HPLC chromatography, chromatofocussing, selectiveprecipitation with such substances as ammonium sulfate; and others (see,e.g., Scopes, Protein Purification: Principles and Practice (1982); U.S.Pat. No. 4,673,641; Ausubel et al. supra; and Sambrook et al., supra).For example, the target protein can be purified using a standardanti-target antibody column Ultrafiltration and diafiltrationtechniques, in conjunction with protein concentration, are also useful Apreferred method of purification utilizes Ni-NTA agarose (Qiagen).

The expressed protein can be purified by standard chromatographicprocedures to yield a purified, biochemically active protein. Theactivity of any of the peptides provided herein can be routinelyconfirmed by the assays provided herein such as those which assay ATPaseactivity or microtubule binding activity. Biologically active targetprotein is useful for identifying modulators of target protein orfragments thereof and kinesin superfamily members using in vitro assayssuch as microtubule gliding assays, ATPase assays (Kodama et al., J.Biochem. 99:1465-1472 (1986); Stewart et al., Proc. Nat'l Acad. Sci. USA90:5209-5213 (1993)), and binding assays including microtubule bindingassays (Vale et al., Cell 42:39-50 (1985)), as described in detailbelow.

A. Purification of HsKif9 from Recombinant Bacteria

Recombinant proteins are expressed by transformed bacteria in largeamounts, typically after promoter induction, but expression can beconstitutive. Promoter induction with IPTG is a preferred method ofexpression. Bacteria are grown according to standard procedures in theart. Fresh or frozen bacteria cells are used for isolation of protein.Alternatively, it is possible to purify HsKif9 from bacteria periplasm.

Alternatively, it is possible to purify HsKif9 from bacteria periplasm.After HsKif9 is exported into the periplasm of the bacteria, theperiplasmic fraction of the bacteria can be isolated by cold osmoticshock in addition to other methods known to skill in the art. To isolaterecombinant proteins from the periplasm, the bacterial cells arecentrifuged to form a pellet. The pellet is resuspended in a buffercontaining 20% sucrose. To lyse the cells, the bacteria are centrifugedand the pellet is resuspended in ice-cold 5 mM MgSO₄ and kept in an icebath for approximately 10 minutes. The cell suspension is centrifugedand the supernatant decanted and saved. The recombinant proteins presentin the supernatant can be separated from the host proteins by standardseparation techniques well known to those of skill in the art.

Suitable purification schemes for some specific kinesins are outlined inU.S. Ser. No. 09/295,612, filed Apr. 20, 1999, hereby expresslyincorporated herein in its entirety for all purposes.

B. Standard Protein Separation Techniques For Purifying HsKif9

Solubility Fractionation

Often as an initial step, particularly if the protein mixture iscomplex, an initial salt fractionation can separate many of the unwantedhost cell proteins (or proteins derived from the cell culture media)from the recombinant protein of interest. The preferred salt is ammoniumsulfate. Ammonium sulfate precipitates proteins by effectively reducingthe amount of water in the protein mixture. Proteins then precipitate onthe basis of their solubility. The more hydrophobic a protein is, themore likely it is to precipitate at lower ammonium sulfateconcentrations. A typical protocol includes adding saturated ammoniumsulfate to a protein solution so that the resultant ammonium sulfateconcentration is between 20-30%. This concentration will precipitate themost hydrophobic of proteins. The precipitate is then discarded (unlessthe protein of interest is hydrophobic) and ammonium sulfate is added tothe supernatant to a concentration known to precipitate the protein ofinterest. The precipitate is then solubilized in buffer and the excesssalt removed if necessary, either through dialysis or diafiltration.Other methods that rely on solubility of proteins, such as cold ethanolprecipitation, are well known to those of skill in the art and can beused to fractionate complex protein mixtures.

Size Differential Filtration

The molecular weight of HsKif9 can be used to isolated it from proteinsof greater and lesser size using ultrafiltration through membranes ofdifferent pore size (for example, Amicon or Millipore membranes). As afirst step, the protein mixture is ultrafiltered through a membrane witha pore size that has a lower molecular weight cut-off than the molecularweight of the protein of interest. The retentate of the ultrafiltrationis then ultrafiltered against a membrane with a molecular cut offgreater than the molecular weight of the protein of interest. Therecombinant protein will pass through the membrane into the filtrate.The filtrate can then be chromatographed as described below.

Column Chromatography

HsKif9 can also be separated from other proteins on the basis of itssize, net surface charge, hydrophobicity, and affinity for ligands. Inaddition, antibodies raised against proteins can be conjugated to columnmatrices and the proteins immunopurified; All of these methods are wellknown in the art. It will be apparent to one of skill thatchromatographic techniques can be performed at any scale and usingequipment from many different manufacturers (e.g., Pharmacia Biotech).

V. Immunological Detection of HsKif9

In addition to the detection of HsKif9 genes and gene expression usingnucleic acid hybridization technology, one can also use immunoassays todetect HsKif9. Immunoassays can be used to qualitatively orquantitatively analyze HsKif9. A general overview of the applicabletechnology can be found in Harlow & Lane, Antibodies: A LaboratoryManual (1988).

A. Antibodies to HsKif9

Methods of producing polygonal and monoclonal antibodies that reactspecifically with HsKif9 are known to those of skill in the art (see,e.g. Coligan, Current Protocols in Immunology (1991); Harlow & Lane,supra; Goding, Monoclonal Antibodies: Principle and Practice (2d ed.1986); and Kohler & Milstein, Nature 256:495-497 (1975). Such techniquesinclude antibody preparation by selection of antibodies from librariesof recombinant antibodies in phage or similar vectors, as well aspreparation of polyclonal and monoclonal antibodies by immunizingrabbits or mice (see, e.g. Huse et al., Science 246:1275-1281 (1989);Ward et al., Nature 341:544546 (1989)).

Humanized forms of mouse antibodies can be generated by linking the CDRregions of non-human antibodies to human constant regions by recombinantDNA techniques. See Queen et al. (1989) Proc. Natl. Acad. Sci. USA86:10029-10033 and PCT Publication WO 90/07861 (each of which isincorporated herein by reference for all purposes).

Human antibodies can be obtained using phage-display methods. See, e.g.,Dower et al. PCT Publication WO 91/17271; McCafferty et al. PCTPublication WO 92/01047. In these methods, libraries of phage areproduced in which members display different antibodies on their outersurfaces. Antibodies are usually displayed as Fv or Fab fragments. Phagedisplaying antibodies with a desired specificity are selected byaffinity enrichment to HsKif9 or fragments thereof. Human antibodiesagainst HsKif9 can also be produced from non-human transgenic mammalshaving transgenes encoding at least a segment of the humanimmunoglobulin locus and an inactivated endogenous immunoglobulin locus.See, e.g., Lonberg et al. PCT Publication WO 93/12227; Kucherlapati PCTPublication WO 91/10741 (each of which is incorporated herein byreference in its entirety for all purposes). Human antibodies can beselected by competitive binding experiments, or otherwise, to have thesame epitope specificity as a particular mouse antibody. Such antibodiesare particularly likely to share the useful functional properties of themouse antibodies. Human polyclonal antibodies can also be provided inthe form of serum from humans immunized with an immunogenic agent.Optionally, such polyclonal antibodies can be concentrated by affinitypurification using HsKif9 as an affinity reagent.

A number of HsKif9 comprising immunogens may be used to produceantibodies specifically reactive with HsKif9. For example, recombinantHsKif9 or a antigenic fragment thereof such as the motor domain, isisolated as described herein. Recombinant protein can be expressed ineukaryotic or prokaryotic cells as described above, and purified asgenerally described above. Recombinant protein is the preferredimmunogen for the production of monoclonal or polyclonal antibodies.Alternatively, a synthetic peptide derived from the sequences disclosedherein and conjugated to a carrier protein can be used an immunogen.Naturally occurring protein may also be used either in pure or impureform. The product is then injected into an animal capable of producingantibodies. Either monoclonal or polyclonal antibodies may be generated,for subsequent use in immunoassays to measure the protein.

Methods of production of polyclonal antibodies are known to those ofskill in the art. An inbred strain of mice (e.g., BALB/C mice) orrabbits is immunized with the protein using a standard adjuvant, such asFreund's adjuvant, and a standard immunization protocol. The animal'simmune response to the immunogen preparation is monitored by taking testbleeds and determining the titer of reactivity to HsKif9. Whenappropriately high titers of antibody to the immunogen are obtained,blood is collected from the animal and antisera are prepared. Furtherfractionation of the antisera to enrich for antibodies reactive to theprotein can be done if desired (see Harlow & Lane, supra).

Monoclonal antibodies may be obtained by various techniques familiar tothose skilled in the art. Briefly, spleen cells from an animal immunizedwith a desired antigen are immortalized, commonly by fusion with amyeloma cell (see Kohler & Milstein, Eur. J: Immunol. 6:511-519 (1976)).Alternative methods of immortalization include transformation withEpstein Barr Virus, oncogenes, or retroviruses, or other methods wellknown in the art. Colonies arising from single immortalized cells arescreened for production of antibodies of the desired specificity andaffinity for the antigen, and yield of the monoclonal antibodiesproduced by such cells may be enhanced by various techniques, includinginjection into the peritoneal cavity of a vertebrate host.Alternatively, one may isolate DNA sequences which encode a monoclonalantibody or a binding fragment thereof by screening a DNA library fromhuman B cells according to the general protocol outlined by Huse et al.,Science 246:1275-1281 (1989).

Monoclonal antibodies and polyclonal sera are collected and titeredagainst the immunogen protein in an immunoassay, for example, a solidphase immunoassay with the immunogen immobilized on a solid support.Typically, polyclonal antisera with a titer of 10⁴ or greater areselected and tested for their cross reactivity against non-HsKif9proteins or even other homologous proteins from other organisms (e.g.,C. elegans unc-104 or human Kif1A), using a competitive bindingimmunoassay. Specific polyclonal antisera and monoclonal antibodies willusually bind with a K_(d) of at least about 0.1 mM, more usually atleast about 1 μM, preferably at least about 0.1 μM or better, and mostpreferably, 0.01 μM or better.

Once HsKif9 specific antibodies are available, HsKf9 can be detected bya variety of immunoassay methods. For a review of immunological andimmunoassay procedures, see Basic and Clinical Immunology (Stites & Terreds., 7th ed. 1991). Moreover, the immunoassays of the present inventioncan be performed in any of several configurations, which are reviewedextensively in Enzyme Immunoassay (Maggio ed., 1980); and Harlow & Lane,supra.

B. Binding Assays

Antibodies can be used for treatment or to identify the presence ofHsKif9 having the sequence identity characteristics as described herein.Additionally, antibodies can be used to identify modulators of theinteraction between the antibody and HsKif9 as further described below.While the following discussion is directed toward the use of antibodiesin the use of binding assays, it is understood that the same generalassay formats such as those described for “non-competitive” or“competitive” assays can be used with any compound which binds to HsKif9such as microtubules or the compounds described in U.S. Pat. No.0.6,207,403.

In a preferred embodiment, HsKif9 is detected and/or quantified usingany of a number of well recognized immunological binding assays (see,e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168).For a review of the general immunoassays, see also Methods in CellBiology Volume 37: Antibodies in Cell Biology (Asai, ed. 1993); Basicand Clinical Immunology (Stites & Terr, eds., 7th ed. 1991).Immunological binding assays (or immunoassays) typically use an antibodythat specifically binds to a protein or antigen of choice (in this casethe HsKif9 or antigenic subsequence thereof). The antibody (e.g.,anti-HsKif9) may be produced by any of a number of means well known tothose of skill in the art and as described above.

Immunoassays also often use a labeling agent to specifically bind to andlabel the complex formed by the antibody and antigen. The labeling agentmay itself be one of the moieties comprising the antibody/antigencomplex. Thus, the labeling agent may be a labeled HsKif9 polypeptide ora labeled anti-HsKif9 antibody. Alternatively, the labeling agent may bea third moiety, such a secondary antibody, that specifically binds tothe antibody/HsKif9 complex (a secondary antibody is typically specificto antibodies of the species from which the first antibody is derived).Other proteins capable of specifically binding immunoglobulin constantregions, such as protein A or protein G may also be used as the labelagent. These proteins exhibit a strong non-immunogenic reactivity withimmunoglobulin constant regions from a variety of species (see generallyKronval et al., J. Immunol. 111:1401-1406 (1973); Akerstrom et al., J.Immunol. 135:2589-2542 (1985)). The labeling agent can be modified witha detectable moiety, such as biotin, to which another molecule canspecifically bind, such as streptavidin. A variety of detectablemoieties are well known to those skilled in the art.

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, preferably from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,antigen, volume of solution, concentrations, and the like. Usually, theassays will be carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 4° C. to 40° C.

Non-Competitive Assay Formats

Immunoassays for detecting HsKif9 in samples may be either competitiveor noncompetitive. Noncompetitive immunoassays are assays in which theamount of antigen is directly measured. In one preferred “sandwich”assay, for example, the anti-HsKif9 antibodies can be bound directly toa solid substrate on which they are immobilized. These immobilizedantibodies then capture HsKif9 present in the test sample. HsKif9 isthus immobilized is then bound by a labeling agent, such as a secondHsKif9 antibody bearing a label. Alternatively, the second antibody maylack a label, but it may, in turn, be bound by a labeled third antibodyspecific to antibodies of the species from which the second antibody isderived. The second or third antibody is typically modified with adetectable moiety, such as biotin, to which another moleculespecifically binds, e.g., streptavidin, to provide a detectable moiety.

Competitive Assay Formats

In competitive assays, the amount of HsKif9 present in the sample ismeasured indirectly by measuring the amount of a known, added(exogenous) HsKif9 displaced (competed away) from an anti-HsKif9antibody by the unknown HsKif9 present in a sample. In one competitiveassay, a known amount of HsKif9 is added to a sample and the sample isthen contacted with an antibody that specifically binds to HsKif9. Theamount of exogenous HsKif9 bound to the antibody is inverselyproportional to the concentration of HsKif9 present in the sample. In aparticularly preferred embodiment, the antibody is immobilized on asolid substrate. The amount of HsKif9 bound to the antibody may bedetermined either by measuring the amount of HsKif9 present in aHsKif9/antibody complex, or alternatively by measuring the amount ofremaining uncomplexed protein. The amount of HsKif9 may be detected byproviding a labeled HsKif9 molecule.

A hapten inhibition assay is another preferred competitive assay. Inthis assay the known HsKif9, is immobilized on a solid substrate. Aknown amount of anti-HsKif9 antibody is added to the sample, and thesample is then contacted with the HsKif9. The amount of anti-HsKif9antibody bound to the known immobilized HsKif9 is inversely proportionalto the amount of HsKif9 present in the sample. Again, the amount ofimmobilized antibody may be detected by detecting either the immobilizedfraction of antibody or the fraction of the antibody that remains insolution. Detection may be direct where the antibody is labeled orindirect by the subsequent addition of a labeled moiety thatspecifically binds to the antibody as described above.

Cross-Reactivity Determinations

Immunoassays in the competitive binding format can also be used forcrossreactivity determinations. For example, a protein at leastpartially encoded by SEQ D NO:2 can be immobilized to a solid support.Proteins (e.g., C. elegans unc-104 or human Kif1A) are added to theassay that compete for binding of the antisera to the immobilizedantigen. The ability of the added proteins to compete for binding of theantisera to the immobilized protein is compared to the ability of HsKif9of SEQ ID NO:2 or SEQ ID NO:4 to compete with itself. The percentcrossreactivity for the above proteins is calculated, using standardcalculations. Those antisera with less than 10% crossreactivity witheach of the added proteins listed above are selected and pooled. Thecross-reacting antibodies are optionally removed from the pooledantisera by immunoabsorption with the added considered proteins, e.g.,distantly related homologues.

The immuno absorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein,thought to be perhaps the protein of this invention, to the immunogenprotein (i.e., HsKif9 of SEQ ID NO:2). In order to make this comparison,the two proteins are each assayed at a wide range of concentrations andthe amount of each protein required to inhibit 50% of the binding of theantisera to the immobilized protein is determined. If the amount of thesecond protein required to inhibit 50% of binding is less than 10 timesthe amount of the protein of SEQ ID NO:2 or SEQ ID NO:4 that is requiredto inhibit 50% of binding, then the second protein is said tospecifically bind to the polyclonal antibodies generated to a HsKif9immunogen.

Other Assay Formats

Western blot (immunoblot) analysis is used to detect and quantify thepresence of HsKif9 in the sample. The technique generally comprisesseparating sample proteins by gel electrophoresis on the basis ofmolecular weight, transferring the separated proteins to a suitablesolid support, (such as a nitrocellulose filter, a nylon filter, orderivatized nylon filter); and incubating the sample with the antibodiesthat specifically bind HsKif9. The anti-HsKif9 antibodies specificallybind to the HsKif9 on the solid support. These antibodies may bedirectly labeled or alternatively may be subsequently detected usinglabeled antibodies (e.g., labeled sheep anti-mouse antibodies) thatspecifically bind to the anti-HsKif9 antibodies.

Other assay formats include liposome immunoassays (LIA), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see Monroe et al., Amer.Clin. Prod Rev. 5:3441 (1986)).

Reduction of Non-Specific Binding

One of skill in the art will appreciate that it is often desirable tominimize non-specific binding in immunoassays. Particularly, where theassay involves an antigen or antibody immobilized on a solid substrateit is desirable to minimize the amount of non-specific binding to thesubstrate. Means of reducing such non-specific binding are well known tothose of skill in the art. Typically, this technique involves coatingthe substrate with a proteinaceous composition. In particular, proteincompositions such as bovine serum albumin (13SA), nonfat powdered milk,and gelatin are widely used with powdered milk being most preferred.

Labels

The particular label or detectable group used in the assay is not acritical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., DYNABEADS™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), colorimetric labels such as colloidalgold or colored glass or plastic beads (e.g., polystyrene,polypropylene, latex, etc.) or other labels that can be detected by massspectroscopy, NMR spectroscopy, or other analytical means known in theart.

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to another molecules (e.g., streptavidin)molecule, which is either inherently detectable or covalently bound to asignal system, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. The ligands and their targets can be used inany suitable combination with antibodies that recognize HsKif9, orsecondary antibodies that recognize anti-HsKif9.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidases, particularlyperoxidases. Fluorescent compounds include fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems which may be used, see U.S. Pat.No. 4,391,904.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink while various conjugated beads appear the color of the bead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

VI. Assays for Modulators of the Target Protein

A. Functional Assays

Assays that can be used to test for modulators of the target proteininclude a variety of in vitro or in vivo assays, e.g., microtubulegliding assays, binding assays such as microtubule binding assays,microtubule depolymerization assays, and ATPase assays (Kodama et al.,J. Biochem. 99: 1465-1472 (1986); Stewart et al., Proc. Natl. Acad. Sci.USA 90: 5209-5213 (1993); Lombillo et al., J. Cell Biol. 128:107-115(1995); Vale et al., Cell 42:39-50 (1985)). Methods of performingmotility assays are well known (see, e.g., Hall et al. (1996) Biophys.J. 371:3467-3476; Turner et al. (1996) Anal. Biochem. 242:20-5; Gitteset al. (1996) Biophys. J. 70:418-29; Shirakawa et al. (1995) J. Exp.Biol. 198:1809-15; Winkelmann et al. (1995) Biophys J. 68:2444-53;Winkelnann et al. (1995) Biophys. J. 68:72S, and the like).

Modulation is tested by screening for candidate agents capable ofmodulating the activity of the target protein comprising the steps ofcombining a candidate agent with the target protein, as above, anddetermining an alteration in the biological activity of the targetprotein. Thus, in this embodiment, the candidate agent should both bindto the target protein (although this may not be necessary), and alterits biological or biochemical activity as defined herein. The methodsinclude both in vitro screening methods and in vivo screening of cellsfor alterations in cell cycle distribution, cell viability, or for thepresence, morphology, activity, distribution, or amount of mitoticspindles, as are generally outlined above.

A preferred assay for high throughput screening is an ATPase assay withcalorimetric detection, e.g., malachite green for end-point detection orcoupled PK/LDH for continuous rate monitoring. An exemplary ATPaseactivity assay utilizes 0.3M PCA (perchloric acid) and malachite greenreagent (8.27 mM sodium molybdate II, 0.33 mM malachite green oxalate,and 0.8 mM triton X-100). To perform the assay, 10 μL, of reaction isquenched in 90 μL of cold 0.3 M PCA. Phosphate standards are used sodata can be converted to mM inorganic phosphate released. When allreactions and standards have been quenched in PCA, 100 μL of malachitegreen reagent is added to the relevant wells in e.g., a microtiterplate. The mixture is developed for 10-15 minutes and the plate is readat an absorbance of 650 nm. If phosphate standards were used, absorbancereadings can be converted to mM Pi and plotted over time. Additionally,ATPase assays known in the art include the luciferase assay.

Another exemplary assay can be performed using the following twospecific solutions. Solution A contains 1 mM ATP, 2 mMphosphoenolpyruvate in a working buffer (25 mM Pipes pH 6.8), 2 mMMgCl₂, 1 mM EGTA, 1 mM DTT, 5 μM taxol, 25 ppm antifoam, pH 6.8.Solution B contains 0.6 mM NADH, 0.2 mg/ml BSA, 1:100 dilution of PK/LDHmixture from Sigrna, 200 μg/ml microtubules, 100 nM HsKif9 (i.e., about2.5 μg/ml). To initiate the experiment, 1 μl of DMSO stock of testcompound μs added to each well of the bottom row of a 96-well half areaplate. Control wells contain only DMSO alone. 50 μl of solution A isthen added to each well. The solutions are mixed by repeated pipetting,followed by a series of dilutions by repeated transferring of 50 μl ofsolution between rows. The reaction is initiated by adding 50 μl ofsolution B. The plate is then inserted in the reader and absorbance at340 nM is monitored for five minutes. The observed rate for 50 μLSolution A+50 μL Solution B in a half-area plate should be about 100mOD/min. Optionally, a series of dilutions is made and absorbancesimilarly measured. Similar procedures can be used to study theinhibitory effect of a test agent on the basal (i.e., notmicrotubule-dependent) ATPase of HsKif9. In these assays, microtubulesare omitted from Solution B and HsKif9 concentration is increased to atleast 2 mM.

Such assays can be used to test for the activity of HsKif9 isolated fromendogenous sources or recombinant sources. Furthermore, such assays canbe used to test for modulators of HsKif9. Modulators can increase ordecrease activity of HsKif9.

In a preferred embodiment, molecular motor activity is measured by themethods disclosed in Ser. No. 09/314,464, filed May 18, 1999, entitled“Compositions and assay utilizing ADP or phosphate for detecting proteinmodulators”, which is incorporated herein by reference in its entirety.More specifically, this assay detects modulators of any aspect of akinesin motor function ranging from interaction with microtubules tohydrolysis of ATP. ADP or phosphate is used as the readout for proteinactivity.

There are a number of enzymatic assays known in the art which use ADP asa substrate. For example, kinase reactions such as pyruvate kinases areknown. See, Nature 78:632 (1956) and Mol. Pharmacol. 6:31 (1970). Thisis a preferred method in that it allows the regeneration of ATP. In oneembodiment, the level of activity of the enzymatic reaction isdetermined directly. In a preferred embodiment, the level of activity ofthe enzymatic reaction which uses ADP as a substrate is measuredindirectly by being coupled to another reaction. For example, in oneembodiment, the method further comprises a lactate dehydrogenasereaction under conditions which normally allow the oxidation of NADH,wherein said lactate dehydrogenase reaction is dependent on the pyruvatekinase reaction. Measurement of enzymatic reactions by coupling is knownin the art Furthermore, there are a number of reactions which utilizephosphate. Examples of such reactions include a purine nucleosidephosphorylase reaction. This reaction can be measured directly orindirectly. A particularly preferred embodiments utilizes the pyruvatekinase/lactate dehydrogenase system.

In one embodiment, the detection of the ADP or phosphate proceedsnon-enzymatically, for example, by binding or reacting the ADP orphosphate with a detectable compound. For example, phosphomolybdatebased assays may be used which involve conversion of free phosphate to aphosphomolybdate complex. One method of quantifying the phosphomolybdateis with malachite green. Alternatively, a fluorescently labeled form ofa phosphate binding protein, such as the E. coli phosphate bindingprotein, can be used to measure phosphate by a shift in itsfluorescence.

In addition, target protein activity can be examined by determiningmodulation of target protein in vitro using cultured cells. The cellsare treated with a candidate agent and the effect of such agent on thecells is then determined either directly or by examining relevantsurrogate markers. For example, characteristics such as mitotic spindlemorphology and cell cycle distribution can be used to determine theeffect.

Thus, in a preferred embodiment, the methods comprise combining a targetprotein and a candidate agent, and determining the effect of thecandidate agent on the target protein. Generally a plurality of assaymixtures are run in parallel with different agent concentrations toobtain a differential response to the various concentrations. Typically,one of these concentrations serves as a negative control, i.e., at zeroconcentration or below the level of detection.

As will be appreciated by those in the art, the components may be addedin buffers and reagents to assay target protein activity and giveoptimal signals. Since the methods allow kinetic measurements, theincubation periods can be optimized to give adequate detection signalsover the background.

In a preferred embodiment, an antifoam or a surfactant is included inthe assay mixture. Suitable antifoams include, but are not limited to,antifoam 289 (Sigma). Suitable surfactants include, but are not limitedto, Tween, Tritons, including Triton X-100, saponins, andpolyoxyethylene ethers. Generally, the antifoams, detergents, orsurfactants are added at a range from about 0.01 ppm to about 10 ppm.

A preferred assay design is also provided. In one aspect, the inventionprovides a multi-time-point (kinetic) assay, with at least two datapoints being preferred In the case of multiple measurements, theabsolute rate of the protein activity can be determined.

B. Binding Assays

In a preferred embodiment, the binding of the candidate agent isdetermined through the use of competitive binding assays. In thisembodiment, the competitor is a binding moiety known to bind to thetarget protein, such as an antibody, peptide, binding partner, ligand,etc. Under certain circumstances, there may be competitive binding asbetween the candidate agent and the binding moiety, with the bindingmoiety displacing the candidate agent.

Competitive screening assays may be done by combining the target proteinand a drug candidate in a first sample. A second sample comprises acandidate agent, the target protein and a compound that is known tomodulate the target protein. This may be performed in either thepresence or absence of microtubules, The binding of the candidate agentis determined for both samples, and a change, or difference in bindingbetween the two samples indicates the presence of an agent capable ofbinding to the target protein and potentially modulating its activity.That is, if the binding of the candidate agent is different in thesecond sample relative to the first sample, the candidate agent iscapable of binding to the target protein.

In one embodiment, the candidate agent is labeled. Either the candidateagent, or the competitor, or both, is added first to the target proteinfor a time sufficient to allow binding. Incubations may be performed atany temperature which facilitates optimal activity, typically between 4and 40° C. Incubation periods are selected for optimum activity, but mayalso be optimized to facilitate rapid high throughput screening.Typically between 0.1 and 1 hour will be sufficient. Excess reagent isgenerally removed or washed away. The second component is then added,and the presence or absence of the labeled component is followed, toindicate binding.

In a preferred embodiment, the competitor is added first, followed bythe candidate agent. Displacement of the competitor is an indication thecandidate agent is binding to the target protein and thus is capable ofbinding to, and potentially modulating, the activity of the targetprotein. In this embodiment, either component can be labeled. Thus, forexample, if the competitor is labeled, the presence of label in the washsolution indicates displacement by the agent. Alternatively, if thecandidate agent is labeled, the presence of the label on the supportindicates displacement.

In an alternative embodiment, the candidate agent is added first, withincubation and washing, followed by the competitor. The absence ofbinding by the competitor may indicate the candidate agent is bound tothe target protein with a higher affinity. Thus, if the candidate agentis labeled, the presence of the label on the support, coupled with alack of competitor binding, may indicate the candidate agent is capableof binding to the target protein.

C. Candidate Agents

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 100 and less than about 2,500 daltons.Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof. Particularly preferred are peptides.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. In a preferred embodiment,the candidate agents are organic chemical moieties, a wide variety ofwhich are available in the literature. Combinatorial libraries can beproduced for many types of compounds that can be synthesized instep-by-step fashion. Such compounds include polypeptides, proteins,nucleic acids, beta-turn mimetics, polysaccharides, phospholipids,hormones, prostaglandins, steroids, aromatic compounds, heterocycliccompounds, benzodiazepines, oligomeric N-substituted glycines andoligocarbamates. Large combinatorial libraries of compounds can beconstructed using the methods described in PCT Publication No. WO95/12608, WO 93/06121, 94/08051, WO 95/35503 and WO 95/30642 (each ofwhich is incorporated herein by reference in its entirety for allpurposes). Peptide libraries can also be generated by phage displaymethods. See, PCT Publication No. WO 91/18980. Compounds to be screenedcan also be obtained from governmental or private sources, including,e.g., the National Cancer Institute's (NCI) Natural Product Repository,Bethesda, Md.; the NCI Open Synthetic Compound Collection, Bethesda,Md.; NCI's Developmental Therapeutics Program, and the like.

D. Other Assay Components

The assays provided utilize target protein as defined herein. In oneembodiment, portions of target protein are utilized; in a preferredembodiment, portions having target protein activity as described hereinare used. In addition, the assays described herein may utilize eitherisolated target proteins or cells or animal models comprising the targetproteins.

A variety of other reagents may be included in the screening assays.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc which may be used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Also,reagents that otherwise improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.,may be used. The mixture of components may be added in any order thatprovides for the requisite binding.

VII. Applications

The methods of the invention are used to identify compounds useful inthe treatment of cellular proliferation diseases. Disease states whichcan be treated by the methods and compositions provided herein include,but are not limited to, cancer (further discussed below), autoimmunedisease, arthritis, graft rejection, inflammatory bowel disease,proliferation induced after medical procedures, including, but notlimited to, surgery, angioplasty, and the like. It is appreciated thatin some cases the cells may not be in a hyper or hypo proliferationstate (abnormal state) and still require treatment For example, duringwound healing, the cells may be proliferating “normally”, butproliferation enhancement may be desired. Similarly, as discussed above,in the agriculture arena, cells may be in a “normal” state, butproliferation modulation may be desired to enhance a crop by directlyenhancing growth of a crop, or by inhibiting the growth of a plant ororganism which adversely affects the crop. Thus, in one embodiment, theinvention herein includes application to cells or individuals afflictedor impending affliction with any one of these disorders or states.

The compositions and methods provided herein are particularly deemeduseful for the treatment of cancer including solid tumors such as skin,breast, brain, cervical carcinomas, testicular carcinomas, etc. Moreparticularly, cancers that may be treated by the compositions andmethods of the invention include, but are not limited to: Cardiac:sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma),myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogeniccarcinoma (squamous cell, undifferentiated small cell, undifferentiatedlarge cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchialadenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma;Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma,leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma,leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma,glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel(adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma,leiomyoma); Genitourinarv tract: kidney (adenocarcinoma, Wilm's tumor[nephroblastoma], lymphoma, leukemia), bladder and urethra (squamouscell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver:hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenicsarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma,chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cellsarcoma), multiple myeloma, malignant giant cell tumor chordoma,osteochronfroma (osteocartilaginous exostoses), benign chondroma,chondroblastoma, chondromyxofibroma, osteoid osteoma and giant celltumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma,osteitis deformans), meninges (meningioma, meningiosarcoma,gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma,germnuoma [pinealoma], glioblastoma multiform, oligodendroglioma,schwannoma, retinoblastoma, congenital tumors), spinal cordneurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus(endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervicaldysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinomamucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecalcell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignantteratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma,adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma,squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma],fallopian tubes (carcinoma); Hematologic: blood (myeloid leukemia [acuteand chronic], acute lymphoblastic leukemia, chronic lymphocyticleukemia, myeloproliferative diseases, multiple myeloma, myelodysplasticsyndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignantlymphoma]; Skin: malignant melanoma, basal cell carcinoma, squamous cellcarcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma,dermatofibroma, keloids, psoriasis; and Adrenal glands: neuroblastomaThus, the term “cancerous cell” as provided herein, includes a cellafflicted by any one of the above identified conditions.

Accordingly, the compositions of the invention are adminstered to cells.By “administered” herein is meant administration of a therapeuticallyeffective dose of the candidate agents of the invention to a cell eitherin cell culture or in a patient. By “therapeutically effective dose”herein is meant a dose that produces the effects for which it isadministered. The exact dose will depend on the purpose of thetreatment, and will be ascertainable by one skilled in the art usingknown techniques. As is known in the art, adjustments for systemicversus localized delivery, age, body weight, general health, sex, diet,time of administration, drug interaction and the severity of thecondition may be necessary, and will be ascertainable with routineexperimentation by those skilled in the art. By “cells” herein is meantalmost any cell in which mitosis or meiosis can be altered.

A “patient” for the purposes of the present invention includes bothhumans and other animals, particularly mammals, and other organisms.Thus the methods are applicable to both human therapy and veterinaryapplications. In the preferred embodiment the patient is a mammal, andin the most preferred embodiment the patient is human.

Candidate agents having the desired pharmacological activity may beadministered in a physiologically acceptable carrier to a patient, asdescribed herein. Depending upon the manner of introduction, thecompounds may be formulated in a variety of ways as discussed below. Theconcentration of therapeutically active compound in the formulation mayvary from about 0.1-100 wt. %. The agents may be administered alone orin combination with other treatments, i.e., radiation, or otherchemotherapeutic agents.

In a preferred embodiment, the pharmaceutical compositions are in awater soluble form, such as pharmaceutically acceptable salts, which ismeant to include both acid and base addition salts.

The pharmaceutical compositions can be prepared in various forms, suchas granules, tablets, pills, suppositories, capsules, suspensions,salves, lotions and the like. Pharmaceutical grade organic or inorganiccarriers and/or diluents suitable for oral and topical use can be usedto make up compositions containing the therapeutically-active compounds.Diluents known to the art include aqueous media, vegetable and animaloils and fats. Stabilizing agents, wetting and emulsifying agents, saltsfor varying the osmotic pressure or buffers for securing an adequate pHvalue, and skin penetration enhancers can be used as auxiliary agents.The pharmaceutical compositions may also include one or more of thefollowing: carrier proteins such as serum albumin; buffers; fillers suchas microcrystalline cellulose, lactose, corn and other starches; bindingagents; sweeteners and other flavoring agents; coloring agents; andpolyethylene glycol. Additives are well known in the art, and are usedin a variety of formulations.

The administration of the candidate agents of the present invention canbe done in a variety of ways as discussed above, including, but notlimited to, orally, subcutaneously, intravenously, intranasally,transdermally, intraperitoneally, intramuscularly, intrapulmonary,vaginally, rectally, or intraocularly. In some instances, for example,in the treatment of wounds and inflammation, the candidate agents may bedirectly applied as a solution or spray.

One of skill in the art will readily appreciate that the methodsdescribed herein also can be used for diagnostic applications. Adiagnostic as used herein is a compound or method that assists in theidentification and characterization of a health or disease state inhumans or other animals. More specifically, antibodies whichspecifically bind HsKif9 may be used for the diagnosis of disorderscharacterized by expression of HsKif9 or in assays to monitor patientsbeing treated with HsKif, or agonists, antagonists, or inhibitors ofHsKif9. Diagnostic assays include methods which utilize the antibody anda label to detect HsKif9 in human body fluids or in extracts of cells ortissues. The antibodies may be used with or without modification, andmay be labeled by covalent or non-covalent attachment of a reportermolecules. A wide variety of reporter molecules are known in the art andmay be used.

A variety of protocols for measuring HsKif9, including ELISAs, RIAs, andFACS, are known in the art and provide a basis for diagnosing altered orabnormal levels of HsKif9 expression. Normal or standard values forHsKif9 expression are established by coming body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toHsKif9 under conditions suitable for complex formation. The amount ofstandard complex formation may be quantitated by various methods,preferably by photometric means. Quantities of HsKif9 expressed insubject, control and disease samples from biopsied tissues can becompared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingHsKif9 may be used for diagnostic purposes. The polynucleotides whichmay be used include oligonucleotide sequences, complementary RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which HsKif9 may becorrelated with disease. The diagnostic assay may be used to determineabsence, presence, and excess expression of HsKif9, and to monitorregulation of HsKif9 levels during therapeutic intervention

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences encodingHsKif9 or closely related molecules may be used. The specificity of theprobe, whether it's made from a highly specific region or from a lessspecific region, and the stringency of the hybridization oramplification (maximal, high, intermediate, or low) will determinewhether the probe identifies only naturally occurring sequences encodingHsKif9, allelic variants, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably have at least 50% sequence identity to any of theHsKif9 encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and may be derived from the sequence of SEQID NO:1 or SEQ ID NO:3 or from genomic sequences including promoters,enhancers, and introns of the HsKif9 gene.

Means for producing specific hybridization probes for DNAs encodingHsKif9 include the cloning of polynucleotide sequences encoding HsKif9or derivatives thereof into vectors for the production of mRNA probes.Such vectors are known in the art, are commercially available, and maybe used to synthesize RNA probes in vitro by means of the addition ofthe appropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, by radionuclides such as ³²P or ³⁵S, or by enzymatic labels,such as alkaline phosphatase coupled to the probe via avidin/biotincoupling systems and the like.

In a particular aspect, the nucleotide sequences encoding HsKif9 may beuseful in assays that detect the presence of associated disorders. Thenucleotide sequences encoding HsKif9 may be labeled by standard methodsand added to a fluid or tissue sample from a patient under conditionssuitable for the formation of hybridization complexes. After a suitableincubation period, the sample is washed and the signal is quantitatedand compared with a standard value. If the amount of signal issignificantly altered in comparison to a control sample then thepresence of altered levels of nucleotide sequences encoding HsKif9 inthe sample indicates the presence of the associated disorder. Suchassays may also be used to evaluate the efficacy of a particulartherapeutic treatment regimen in animal studies, in clinical trials, orto monitor the treatment of an individual patient.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding HsKif9 may involve the use of PCR. These oligomersmay be chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding HsKif9, or a fragment of a polynucleotide complementary to thepolynucleotide encoding HsKif9, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection orquantitation of closely related DNA or RNA sequences.

Methods which may be used to quantitate the expression of HsKif9 includeradiolabeling or biotinylating nucleotides, coamplification of a controlnucleic acid, and interpolating results from standard curves. The speedof quantitation of multiple samples may be accelerated by running theassay in an ELISA format where the oligomer of interest is presented invarious dilutions and a spectrophotometer or colorimetric response givesrapid quantitation.

One of skill in the art will readily appreciate that the methodsdescribed herein also can be used for diagnostic applications. Adiagnostic as used herein is a compound or method that assists in theidentification and characterization of a health or disease state inhumans or other animals.

The present invention also provides for kits for screening formodulators of the target protein. Such kits can be prepared from readilyavailable materials and reagents. For example, such kits can compriseany one or more of the following materials: biologically active targetprotein, reaction tubes, and instructions for testing activity of thetarget protein. Preferably, the kit contains biologically active targetprotein. A wide variety of kits and components can be prepared accordingto the present invention, depending upon the intended user of the kitand the particular needs of the user. For example, the kit can betailored for ATPase assays, microtubule gliding assays, or microtubulebinding assays.

The kinesins of the present invention and, in particular, their motordomains can be used for separation of a specific ligand from aheterologous mixture in aqueous solution as described by Stewart, U.S.Pat. No. 5,830,659. In the system discussed by Stewart, a kinesin motordomain is linked to a ligand binding moiety, such as streptavidin. Thechimeric kinesin motor domains are placed into a loading chambercontaining the heterogeneous mixtures which is coupled to a receivingchamber by a channel bearing immobilized, aligned microtubules. Additionof ASP to the loading chamber results in translocation of the kinesinmotor domains, now attached covalently to the receiving chamber. Hence,the ATP-driven motility activity of the kinesin motor domain results inseparation of the desired ligand from the heterogeneous mixture. Stewartfurther teaches that all kinesin motor domains are suitable for use inthe separation system

The kinesins of their invention and, in particular, their motor domainscan also be used in the field of nanotechnology. Molecular motors suchas kinesin have widespread application in the construction of nanoscalemachines. Biomolecular motors have real-world application in theemerging nanotechnological arts. For example, a 1999 NASA studyidentifies multiple applications for nanoscale motors—and kinesin inparticular— in the aerospace field. Kinesin motor domains can be used inthe construction of rotors and other mechanical components (for review,see Limberis and Stewart (2000) Nanotechnology 11:47-51) as well aslight-operated molecular shuttles useful for nanoscale switches andpumps.

Nucleic acids encoding the kinesins of the invention are also useful forinclusion on a GeneChip™ array or the like for use in expressionmonitoring (see U.S. Pat. No. 6,040,138, EP 853,679 and WO 97/27317).Such arrays typically contain oligonucleotide or cDNA probes to allowdetection of large numbers of mRNAs within a mixture. Many of thenucleic acids included in such arrays are from genes or ESTs that havenot been well characterized. Such arrays are often used to compareexpression profiles between different tissues or between differentconditions of the same tissue (healthy vs. diseased or drug-treated vs.control) to identify differentially expressed transcripts. Thedifferentially expressed transcripts are then useful, e.g., fordiagnosis of disease states, or to characterize responses of drugs. Thenucleic acids of the invention can be included on GeneChip™ arrays orthe like together with probes containing a variety of other genes. Thepresent nucleic acids are particularly useful for inclusion inGeneChipa™ arrays for analyzing the cell cycle or proliferation state ofcells. Nucleic acids encoding HsKif9 can be combined with nucleic acidsencoding other kinesin molecules and/or nucleic acids from other geneshaving roles in DNA replication, cell division or other cell cyclefunction. Such arrays are useful for analyzing and diagnosing cells in aproliferating state, and diseases such as cancer characterized bypresence of the same. Such arrays are also useful for analyzingcandidate drugs for roles in modulation of the cell cycle andproliferation. The efficacy of such drugs can be assayed by determiningthe effect of the drug on the expression profile of genes affectingproliferation and the cell cycle.

VIII. EXAMPLES

Assay Protocol

This assay is based on detection of ADP production from a targetprotein's microtubule stimulated ATPase. ATP production is monitored bya coupled enzyme system consisting of pyruvate kinase and lactatedehydrogenase. Under the assay conditions described below, pyruvatekinase catalyzes the conversion of ADP and phosphoenol pyruvate topyruvate and ATP. Lactate dehydrogenase then catalyzes theoxidation-reduction reaction of pyruvate and NADH to lactate and NAD+.Thus, for each molecule of ADP produced, one molecule of NADH isconsumed. The amount of NADH in the assay solution is monitored bymeasuring light absorbance at a wavelength of 340 nm.

The final 25 μl assay solution consists of the following: 5 μg/ml targetprotein, 30 U/ml microtubules, 5 μM Taxol, 0.8 mM NADH, 1.5 mMphosphoenol pyruvate, 3.5 U/ml pyruvate kinase, 5 U/ml lactatedehydrogenase, 25 mM Pipes/KOH pH 6.8, 2 mM MgCl₂, 1 mM EGTA, 1 mM MDTT,0.1 mg/ml BSA, 0.001% antifoam 289, and 1 mM ATP.

Potential candidate agents are dissolved in DMSO at a concentration ofabout 1 mg/ml and 0.5 μl of each chemical solution is dispensed into asingle well of a clear 384 well plate. Each of the 384 wells are thenfilled with 20 μl of a solution consisting of all of the assaycomponents described above except for ATP. The plate is agitated at ahigh frequency. To start the assay, 5 μl of a solution containing ATP isadded t6 each well. The plate is agitated and the absorbance is read at340 nm over various time intervals. The assay is run at roomtemperature.

The assay components and the performance of the assay are optimizedtogether to match the overall read time with the rate of the targetprotein's ADP production. The read time should be long enough for therate of NADH consumption to reach steady state beyond an initial lagtime of several seconds.

ATPase Assay Protocol

2 μg/ml of the target protein is assayed for its microtubule-stimulatedATPase activity in a reaction buffer composed of 40 μM MES/KOH pH 6.8, 2mM MgCl₂, 1 mM DTT, 1 mM EGTA, 100 μM ATP, 10 μM paclitaxel 0.1 mg/mlBSA, 0.5 mM NADH, 1.5 mM phosphoenolpyruvate, lactatedehydrogenase/pyruvate kinase mix (Sigma, diluted 1:200 v/v final) andvaried amounts of microtubules (from 50 μg/ml to 24 ng/ml). The reactionprogress is followed over time by monitoring absorbance of the reactionmixture at 340 nm using a microtiter plate reader (SpectraMAX340,Molecular Devices, Inc.). The rate of the absorbance change is convertedto μM NADH oxidized per second by referencing it to a set of standardNADH solutions of known concentrations. In this coupled ATPase assay,conversion of one NADH to NAD+ reports appearance of one ADP molecule,and as such, reports a single turnover of the target molecule ATPase.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety.

1. An isolated nucleic acid sequence encoding a microtubule motorprotein, wherein the motor protein has the following properties: (i) theprotein's activity includes microtubule stimulated ATPase activity; and(ii) the protein has a sequence that has greater than 90% amino acidsequence identity to SEQ ID NO:2 or SEQ ID NO:4 as measured using asequence comparison algorithm.
 2. An isolated nucleic acid sequence ofclaim 1, wherein the protein specifically binds to polyclonal antibodiesgenerated against a protein comprising SEQ ID NO:2 or SEQ ID NO:4.
 3. Anisolated nucleic acid sequence of claim 1, wherein the nucleic acidencodes SEQ ID NO:2 or SEQ ID NO:4.
 4. An isolated nucleic acid sequenceof claim 1, wherein the nucleic acid has a nucleotide sequence of SEQ IDNO:1 or SEQ ID NO:3.
 5. An isolated nucleic acid sequence of claim 1,wherein the nucleic acid selectively hybridizes under stringenthybridization conditions to SEQ ID NO:1 or SEQ D NO:3.
 6. An expressionvector comprising a nucleic acid encoding a microtubule motor protein,wherein the motor protein has the following properties: (i) theprotein's activity includes microtubule stimulated ATPase activity; and(ii) the protein has a sequence that has greater than 90% amino acidsequence identity to SEQ ID NO:2 or SEQ ID NO:4 as measured using asequence comparison algorithm.
 7. A host cell transfected with thevector of claim
 6. 8. An isolated microtubule motor protein, wherein theprotein has greater than 90% amino acid sequence identity to SEQ ID NO:2or SEQ ID NO:4 as measured using a sequence comparison algorithm.
 9. Anisolated protein of claim 8, wherein the protein specifically binds topolyclonal antibodies generated against a protein comprising SEQ ID NO:2or SEQ ID NO:4.
 10. An isolated protein of claim 8, wherein the proteinis SEQ ID NO:2 or SEQ ID NO:4.
 11. An isolated protein of claim 10,wherein the protein has an amino acid sequence of SEQ ID NO:2.
 12. Anisolated protein of claim 10, wherein the protein has an amino acidsequence of SEQ ID NO:4.
 13. A method for screening for modulators ofHsKif9, the method comprising the steps of: (i) providing biologicallyactive HsKif9, wherein has the following properties: (i) activityincluding microtubule stimulated ATPase activity, and (ii) sequence thathas greater than 90% amino acid sequence identity to SEQ ED NO:2 or SEQID NO:4 as measured using a sequence comparison algorithm; (ii)contacting biologically active HsKif9 with a candidate agent in a testand control concentration; and (iii) assaying for the level of HsKif9activity, wherein the HsKif9 activity is selected from the groupconsisting of binding activity or ATPase activity, and wherein a changein activity between the test and control concentration indicates amodulator.
 14. A method of claim 13, wherein the screening occurs in amulti-well plate as part of a high-throughput screen.
 15. A method ofclaim 13, wherein the biologically active HsKif9 comprises an amino acidsequence of SEQ D NO:4.
 16. A compound that modulates HsKif9, whereinsaid compound is identified using the method of claim
 13. 17. Anisolated nucleic acid comprising a sequence which has greater than 80%sequence identity with nucleotide SEQ ID NO:1 or SEQ ID NO:3.